Cationic poly (amino acids) and uses thereof

ABSTRACT

The present invention provides an efficient delivery system for a nucleic acid, more specifically, a cationic poly(amino acid) that has a side chain having a plurality of different amine functional groups in a moiety including a cationic group and that has a hydrophobic group introduced into part of the side chain, and a polyion complex (PIC) of the poly(amino acid) and an oligo- or polynucleotide.

TECHNICAL FIELD

The present invention relates to a cationic poly(amino acid) that has ahydrophobic group introduced into part of a side chain. Morespecifically, the present invention relates to a cationic poly(aminoacid) that has a side chain having a plurality of different aminefunctional groups in a moiety including a cationic group and that has ahydrophobic group introduced into part of the side chain, and a polyioncomplex (PIC) of the poly(amino acid) and an oligo- or polynucleotide.

BACKGROUND ART

The application of siRNA to medical treatments is increasingly expectedbecause the siRNA can knock down target mRNA specifically andeffectively. However, the development of an effective delivery system isindispensable to apply the siRNA to medical treatments. In recent years,it has been clarified that a therapeutic effect of naked siRNA onage-related macular degeneration (CNV) through its intraocularadministration under a clinical trial does not result from asequence-specific gene knockdown effect induced by siRNA but resultsfrom a non-sequence-specific effect via recognition by Toll-likereceptor-3 (TLR-3) on cell surface. It has been considered important todevelop a carrier that is stable outside cells and is capable ofaccurately delivering siRNA into the cells in any of in vivo siRNAapplications.

Poly(L-lysine) or polyethylene imine, which has been long known as acationic polymer for forming a polyion complex (PIC) with DNA tointroduce a gene into eukaryotic cells and expressing the gene, has aproblem in that the compound does not exhibit very high gene expressionefficiency or exhibits high toxicity on cells, for example. A widevariety of cationic polymers have been provided in order to solve suchproblem. For example, a poly(L-lysine) derivative in which a hydrophilicgroup (e.g., polyethylene glycol) and a hydrophobic group (e.g.,palmitoyl) have been introduced via an ε-amino group of poly(L-lysine)forms a vesicle in the presence of cholesterol in an aqueous medium andthe vesicle aggregates gene-containing plasmid DNA to form a stablecomplex (Patent Document 1). Further, a PIC formed of plasmid DNA with acopolymer derivative whose cation charge and disulfide crosslink densityhave been modulated by the thiolation of an ε-amino group ofpoly(L-lysine) in a poly(L-lysine)-poly(ethylene glycol) copolymer showshigh stability in an extracellular medium and effectively releases theDNA in an intracellular compartment (Non Patent Document 1, some of theinventors of the present invention are coauthors of Non Patent Document1). Further, the inventors of the present invention have confirmed, aspart of such research, that, whenpoly(N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide (PAsp(DET))) having anethylenediamine structure in a side chain and a block copolymerincluding the PAsp(DET) as one block component of the block copolymerare produced, such polymers show low cytotoxicity and introduce plasmidDNA into cells with high efficiency to express a gene incorporated intothe DNA efficiently (see Non Patent Document 2, Patent Document 2, andPatent Document 3).

PRIOR ART DOCUMENT Patent Documents

-   [Patent Document 1] WO 99/61512-   [Patent Document 2] WO 2006/085664 A1-   [Patent Document 3] WO 2007/099660 A1

Non Patent Documents

-   [Non Patent Document 1] K. Mihata et al., J. Am. Chem. Soc. 2004,    126, 2355-2361-   [Non Patent Document 2] K. Miyata et al., J. Am. Chem. Soc. 2008,    130, 16287-16294

SUMMARY OF INVENTION Problem to be Solved by the Invention

PAsp(DET) and a block copolymer thereof disclosed in Patent Documents 2and 3 exhibit low toxicity and high gene introduction efficiency ontarget cells to which a nucleic acid molecule is to be delivered, but asmentioned above, may not necessarily form a PIC having high stabilityunder a physiological condition with a low molecular weight nucleic acidsuch as siRNA, which is increasingly expected for its application tomedical treatments. Thus, an object of the present invention is toprovide a synthetic polymer material which may serve as a carrierexhibiting higher stability with a low molecular weight nucleic acidsuch as siRNA under a physiological condition and exhibiting lowtoxicity and high gene introduction efficiency on target cells to whicha nucleic acid molecule is to be delivered.

Means for Solving the Problem

This time, the inventors of the present invention have found that acationic poly (amino acid) that has a side chain having a plurality ofdifferent amine functional groups in a moiety including a cationic groupand that has a hydrophobic group introduced into part of the side chainexhibits no toxicity or little toxicity on target cells to which anucleic acid molecule is to be delivered, and can form a stable PIC witha low molecular weight nucleic acid such as siRNA as well. The inventorshave also found that the thus formed PIC forms a relativelymonodispersed stable associate having an average diameter of ahundred-odd nm in an aqueous medium under a given condition, isincorporated into target cells with high efficiency, and exerts aneffect inherently possessed by siRNA.

According to the present invention, the problem is solved by providing apoly(amino acid) derivative, which is represented by each of thefollowing formula (1) and formula (2):

-   where: R¹ represents a hydroxyl group, an unsubstituted or    substituted linear or branched alkyloxy group having 1 to 12 carbon    atoms, an unsubstituted or substituted linear or branched alkenyloxy    group having 2 to 12 carbon atoms, an unsubstituted or substituted    linear or branched alkynyloxy group having 1 to 12 carbon atoms, or    an unsubstituted or substituted linear or branched alkyl-substituted    imino group having 1 to 12 carbon atoms;-   R² represents a hydrogen atom, an unsubstituted or substituted    linear or branched alkyl group having 1 to 12 carbon atoms, or an    unsubstituted or substituted linear or branched alkylcarbonyl group    having 1 to 24 carbon atoms;-   R^(3a) and R^(3b) each independently represent a methylene group or    an ethylene group;-   R^(4a) and R^(4b) are each independently chosen from the same or    different groups in the group consisting of the following groups:

—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i);

—NH—(CH₂)_(p2)—N[—(CH₂)_(q2)—NH₂]₂   (ii);

—NH—(CH₂)_(p3)—N{[—(CH₂)_(q3)—NH₂][—(CH₂)_(q4)—NH]_(r2)H}  (iii); and

—NH—(CH₂)_(p4)—N{—(CH₂)_(q5)—N[—(CH₂)_(q6)—NH₂]₂}₂   (iv),

where: p1 to p4, q1 to q6, and r1 and r2 each independently represent aninteger of 1 to 5;

-   5 to 40% of a total number of the group of each of R^(4a) and R^(4b)    have at least one amino group in which a hydrogen atom is    substituted by an acyl group having a saturated or unsaturated    linear or branched aliphatic hydrocarbon residue having 6 to 27    carbon atoms, or a steroloxycarbonyl group;-   n represents an integer of 30 to 5,000; and-   y represents an integer of 0 to 5,000,-   provided that, when R^(3a) and R^(3b) each represent a methylene    group, y represents an integer smaller than n.

-   where: R², R^(3a), R^(3b), R^(4a), R^(4b), n, and y have the same    meanings as those defined for the formula (1);-   L¹ represents —S—S— or a valence bond;-   L² represents —NH—, —O—, —O(CH₂)_(p1)—NH—, or    -L^(2a)-(CH₂)_(q1)-L^(2b)-, where:-   p1 and q1 each independently represent an integer of 1 to 5; L^(2a)    represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH, or COO; and L^(2b)    represents NH or O;-   R⁵ represents a hydrogen atom or an unsubstituted or substituted    linear or branched alkyl group having 1 to 12 carbon atoms; and m    represents an integer of 30 to 20,000.

Unless otherwise indicated, it should be understood that the groups ormoieties in the formula (1) and formula (2), or groups defined thereforare covalently bonded in the described directionality. In this regard,however, a repetitive unit having a repetition number of n-y and arepetitive unit having a repetition number of y in the formula (1) andformula (2) are arranged in any suitable manner, and any suitablestructure such as a block structure, a random structure, or an alternatestructure is applicable.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram for illustrating the respective structural formulaeof PAsp(DET) (on the left of the figure) and PAsp(DET)-ST manufacturedby a method according to Example 1(1).

FIG. 2 is a photograph as an alternative of a diagram for showing theresults of polyacrylamide gel electrophoresis of a PIC of PAsp(DET)-ST19% and siRNA formed by a method according to Example 1(2).

FIG. 3 is a graph illustrating the evaluation results of the particlediameter and polydispersity index of the PIC of Example 1(2) by dynamiclight scattering. At N/P=5.0, an ST 12% micelle had an average size of171 nm and a polydispersity index (PDI) of 0.144, whereas an ST 19%micelle had an average size of 152 nm and a polydispersity index of0.100.

FIG. 4 is a graph illustrating the results of a knockdown test of anendogenous gene Bcl-2 using the PIC of Example 2(1).

FIG. 5 is a graph illustrating the evaluation results of cytotoxicity inthe knockdown test using the PIC in Example 2(1).

FIG. 6 is a graph illustrating the results of a knockdown test of anendogenous gene VEGF using the PIC in Example 2(2) (evaluation of VEGFexpression).

FIG. 7 is a graph illustrating the results of the knockdown test of theendogenous gene VEGF using the PIC in Example 2(2) (evaluation of VEGFat a protein level).

FIG. 8 is a conceptual diagram illustrating polymer micelle formation ofPEG-SS-PAsp(DET)-ST and siRNA synthesized in Example 3 (1).

FIG. 9 is a graph illustrating the results of an siRNA transfection testusing a complex of PEG-SS-PAsp(DET)-ST and siRNA of Example 3(3).

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention is more specifically described.

Without wishing to be bound by theory, a cationic poly(amino acid) ofthe present invention has a side chain having a plurality of differentamine functional groups in a moiety including a cationic group and thusexhibits pKa's at a plurality of stages. Under a physiological conditionat pH 7.4, the plurality of amine functional groups are each in apartially protonated state and can form a polyion complex (PIC) throughan interaction with a nucleic acid. Further, it can be understood that,when the PIC thus formed is taken up into the endosome (pH 5.5), theprotonation of the cationic poly(amino acid) further proceeds owing to adecrease in pH and the promotion of endosome escape through a buffereffect (or proton sponge effect) alleviates damage on cells. Meanwhile,it is understood that, when a hydrophobic group is introduced into amoiety including a cationic group at a specific ratio, the introductiondoes not adversely affect the buffer effect (or proton sponge effect)and the PIC is stabilized through a hydrophobic interaction, and as aresult, a relatively monodispersed stable associate having an averagediameter of a hundred-odd nm is formed. Further, when the cationicpoly(amino acid) as described above according to the present inventionis used in the form of a block copolymer with a PEG segment in the samemanner as described in Patent Document 3, the block copolymer can form apolymer micelle exhibiting satisfactory retentivity in circulating bloodwhile at least retaining the characteristics possessed by the cationicpoly(amino acid) itself.

The alkyl moiety in a linear or branched alkyl group, alkyl-substitutedimino group, or the like having 1 to 12 carbon atoms, which is definedby the R¹ and R² in the formula (1) and formula (2), may be, forexample, a methyl group, an ethyl group, an n-propyl group, an isopropylgroup, an n-butyl group, an sec-butyl group, a tert-butyl group, ann-hexyl group, a decyl group, and an undecyl group. An alkenyl oralkynyl moiety in an unsubstituted or substituted linear or branchedalkenyloxy group having 2 to 12 carbon atoms, an unsubstituted orsubstituted linear or branched alkynyloxy group having 1 to 12 carbonatoms, or the like may be exemplified by one including a double bond ora triple bond in the alkyl group having 2 or more carbon atoms asexemplified above.

For such group or moiety, a substituent in a “substituted” case may beexemplified by, but not limited to, a C₁₋₆ alkoxy group, an aryloxygroup, an aryl C₁₋₃ oxy group, a cyano group, a carboxyl group, an aminogroup, a C₁₋₆ alkoxycarbonyl group, a C₂₋₇ acylamide group, a tri-C₁₋₆alkyl siloxy group, a siloxy group, or a silylamino group, or may beexemplified by an acetalated formyl group, a formyl group, or a halogensuch as chlorine or fluorine. In this context, for example, theexpression “C₁₋₆” means 1 to 6 carbon atoms and is used with the samemeaning in the following description. In addition, an unsubstituted orsubstituted linear or branched alkyl moiety having 1 to 12 carbon atomsin the unsubstituted or substituted linear or branched alkylcarbonylgroup having 1 to 24 carbon atoms may be selected with reference to theexamples, and an alkyl moiety having 13 or more carbon atoms may be, forexample, a tridecyl group, a tetradecyl group, a pentadecyl group, anonadecyl group, a docosanyl group, and a tetracosyl group.

As for the methylene group or the ethylene group in the definition forR^(3a) and R^(3b), it is understood that, when both of R^(3a) and R^(3b)each represent a methylene group, a repetitive unit involving suchdefinition represents a unit derived from poly(aspartic acid), and therespective units having those groups are present at random. Meanwhile,when both of R^(3a) and R^(3b) each represent an ethylene group, arepetitive unit involving such definition represents a unit derived frompoly(glutamic acid), and in this case, represents a polymer in which yrepresents an integer of 0 or n-y represents an integer of 0. The formerrepresents, for example, a unit derived from poly-α-glutamic acidobtained by the polymerization of glutamic acid γ-benzyl esterN-carboxylic anhydride. Meanwhile, the latter represents, for example, aunit derived from poly-γ-glutamic acid produced by a bacterial strainbelonging to the genus Bacillus bacteria such as Bacillus natto.

The group chosen from the group consisting of:

—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i);

—NH—(CH₂)_(p2)—N[—(CH₂)_(q2)—NH₂]₂   (ii);

—NH—(CH₂)_(p3)—N{[—(CH₂)_(q3)—NH₂][—(CH₂)_(q4)—NH]_(r2)H}  (iii); and

—NH—(CH₂)_(p4)—N{—(CH₂)_(q5)—N[—(CH₂)_(q6)—NH₂]₂}₂   (iv),

which is defined for the group of each of R^(4a) and R^(4b), ispreferably the same group, and p1 to p4 and q1 to q6 each independentlyrepresent preferably 2 or 3, more preferably 2. Meanwhile, r1 and r2each independently represent preferably an integer of 1 to 3.

In the group of each of R^(4a) and R^(4b), a hydrogen atom of at leastone amino group in the group accounting for 5 to 40%, preferably 10 to30%, more preferably 15 to 25% of the total number n-y or y of the groupis substituted by an acyl group having a saturated or unsaturated linearor branched aliphatic hydrocarbon residue having 6 to 27 carbon atoms,or a steroloxycarbonyl group. When the aliphatic hydrocarbon residue issaturated, the residue is equivalent to an alkyl group having 6 to 27carbon atoms and is exemplified by a pentacosyl group, a hexacosylgroup, or a heptacosyl group as well as the alkyl group. The unsaturatedaliphatic hydrocarbon residue corresponds to a group in which 1 to 5carbon-carbon single bonds in a chain of the alkyl group are replaced bycarbon-carbon double bonds. An acyl group (RCO—) having such residue (R)may be exemplified by, but not limited to, lauric acid (or dodecanoicacid), myristic acid (or tetradecanoic acid), palmitic acid (orhexadecanoic acid), palmitoleic acid (or 9-hexadecenoic acid), stearicacid (or octadecanoic acid), oleic acid, linoleic acid, linolenic acid,eleostearic acid (or 9,11,13-octadecatrienoic acid), arachidic acid,arachidonic acid, behenic acid, lignoceric acid, nervonic acid, ceroticacid, or montanic acid.

The sterol used herein means a natural, semisynthetic, or syntheticcompound based on a cyclopentanone hydrophenanthrene ring (C₁₇H₂₈) andderivatives thereof. For example, a natural sterol is exemplified by,but not limited to, cholesterol, cholestanol, dihydrocholesterol, cholicacid, campesterol, or sitosterol. Semisynthetic or synthetic compoundsthereof may be, for example, synthetic precursors of these naturalproducts (as necessary, encompassing a compound in which part or all of,if present, certain functional groups, hydroxy groups have beenprotected with a conventional hydroxy protective group, or a compound inwhich a carboxyl group has been protected with carboxyl protection).Further, the sterol derivative means that, for example, withoutadversely affecting the object of the present invention, a C₁₋₁₂ alkylgroup, a halogen atom such as chlorine, bromine, or fluorine may beintroduced into a cyclopentanone hydrophenanthrene ring, and the ringsystem may be saturated or partially unsaturated. A redidue of thesterol derivative is preferably a group in which a hydrogen atom of ahydroxy group at the 3-position of cholesterol, cholestanol, ordihydroxycholesterol has been removed. The residue is more preferably agroup in which a hydrogen atom of a hydroxy group at the 3-position ofcholesterol has been removed. A sterol of the steroloxycarbonyl group isexemplified by one of an animal or vegetable oil origin such ascholesterol, cholestanol, dihydro cholesterol, cholic acid, campesterol,or sitosterol.

n, which represents the number of repetitive units of an amino acid, mayrepresent 30 to 5,000, and in view of convenience in synthesis, mayrepresent 30 to 300, preferably 60 to 150, more preferably 60 to 100.Further, y may represent 0 to 5,000, and in view of convenience insynthesis, may represent 30 to 300, preferably 60 to 150, morepreferably 60 to 100.

According to the present invention, there can also be provided a blockcopolymer represented by the formula (2). In the formula (2) , R²,R^(3a), R^(3b), R^(4a), R^(4b), n, and y in the formula same meanings asthose defined for the formula (1) and described above.

L¹ represents —S—S— or a valence bond. Meanwhile, L² represents —NH—,—O—, —O(CH₂)_(p1)—NH—, or -L^(2a)-(CH₂)_(q1)-L^(2b)-, where: p1 and q1each independently represent an integer of 1 to 5; L^(2a) representsOCO, OCONH, NHCO, NHCOO, NHCONH, CONH, or COO; and L^(2b) represents NHor O. L¹ and L² need to be combined with each other so that they mayform one linking group. For example, when L² represents —NH—, L¹ doesnot represent —S—S— but a valence bond. Preferred -L¹-L²- is one forminga linking group in the case where L¹ represents —S—S—.

R⁵ represents a hydrogen atom or an unsubstituted or substituted linearor branched alkyl group having 1 to 12 carbon atoms. As examplesthereof, ones described for R¹ are applied. m, which represents thenumber of repetitive units of ethylene glycol (or oxyethylene),represents an integer of 30 to 20,000, preferably 200 to 2,000, morepreferably 500 to 1,000.

The poly(amino acid) represented by the formula (1) may be manufactured,for example, by: subjecting a polyamino acid ester, which ismanufactured by the polymerization of a conventional N-carboxylicanhydride derived from an aspartic acid ester or a glutamic acid ester,to aminolysis using a polyamine corresponding to a polyamine residue ofthe group of each of R^(4a) and R^(4b) to introduce the polyamineresidue into a side chain of the poly(amino acid); and subjecting anamino group of a polyamine moiety thus introduced to a reaction with anappropriate amount of an activated carboxylic acid, which is obtained bythe activation of a carboxyl group of a carboxylic acid corresponding toan acyl group having the aliphatic hydrocarbon residue, if required. Theblock copolymer of the formula (2) may be manufactured in accordancewith a conventional linking method for a polyethylene glycol segment andthe poly(amino acid) manufactured as described above, or may bemanufactured in accordance with the method described in Non PatentDocument 3 including manufacturing a block copolymer that has thepolyamine residue being free from a hydrophobic group and then carryingout a reaction with an activated carboxylic acid. The introduction of ahydrophobic group excluding the acyl group may also be carried out by aconventional reaction of an amino group and an active carbonateesterified product carrying the hydrophobic group.

The polymers of the formula (1) and the formula (2) which may beprovided as described above exhibit the pKa's at a plurality of stages,and hence can form a polyion complex (PIC) with an anionicallychargeable compound under a physiological condition. The anionicallychargeable compound may be a protein, a lipid, a peptide, or a nucleicacid. In particular, a PIC with a nucleic acid is conveniently formed.Hence, hereinafter, a PIC of a nucleic acid and a cationic poly(aminoacid) of the present invention or a block copolymer thereof is describedfor the purpose of simplifying the description.

As described above, the cationic poly(amino acid) of the presentinvention has a hydrophobic group in its side chain, and hence thecationic poly(amino acid) can form a PIC with a small molecular weightnucleic acid as well under a physiological condition to provide a stablevesicle or associate. The nucleic acid capable of providing a PIC inaccordance with the present invention using the cationic poly(aminoacid) of the formula (1) or block copolymer of the formula (2) means apoly- or oligonucleotide including as a basic unit nucleotides formed ofa purine or pyrimidine base, a pentose, and phosphoric acid, andexamples thereof may include oligo- or poly-double-stranded RNA, oligo-or poly-double-stranded DNA, oligo- or poly-single-stranded DNA, andoligo- or poly-single-stranded RNA. Further, oligo- orpoly-double-stranded nucleic acid and oligo- or poly-single-strandednucleic acid in each of which RNA and DNA exist in a mixed state in thesame chain are also included. Further, the nucleotide contained in thenucleic acid may be of natural type or of chemically modifiednon-natural type, or may have added thereto an amino group, a thiolgroup, a fluorescent compound, or any other molecule. The nucleic acidis not limited but may be formed of 4 to 20,000 bases, preferably 10 to10,000 bases, more preferably 18 to 30 bases. Further, in considerationof functions or actions, there may be given plasmid DNA, siRNA, microRNA, shRNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer,and a ribozyme.

As the siRNA, for example, all of those designed for a gene or apolynucleotide of interest by a conventional method may be used. For thechain length of siRNA, a moiety for forming a double strand may have alength of preferably 15 to 50 bases, more preferably 18 to 30 bases, andconventional compounds and all nucleotides having the same actions orfunctions as those compounds are encompassed. Specific examples of thesiRNA may be designed with reference to a gene which may serve as atarget of a gene therapy, but are not limited thereto. Examples of suchgene may include, but not limited to, PKCα related to a disease such asnon-small cell lung carcinoma, BCL-2 related to a disease such asmalignant melanoma, ICAM-1 related to Crohn's disease, HCV related to Ctype hepatitis, TNFα related to rheumatoid arthritis or psoriasis,adenosine AI receptor related to asthma, c-raf kinase related to adisease such as ovary cancer, H-ras related to a disease such aspancreas cancer, c-myc related to coronary artery disease, PKA Riarelated to large bowel cancer, HIV related to AIDS, DNA methyltransferase related to solid cancer, VEGF receptor related to cancer,ribonucleotide reduction enzyme related to kidney cancer, CMV IE 2related to CMV retinitis, MMP-9 related to prostate cancer, TGFβ2related to malignant glioma, CD 49 d related to Multiple Sclerosis,PTP-1B related to diabetes, c-myb related to cancer, EGFR related to adisease such as breast cancer, and mdr1, autotaxin and GLUT-1 generelated to cancer. As for the antisense nucleic acid, those known in theart or all having the same functions or actions as those may be employedas a target for forming a PIC in accordance with the present invention.

Thus, the PIC of the nucleic acid and the compound represented by theformula (1) or the formula (2) as described above is provided as anotheraspect of the present invention. Such PIC of the compound of the formula(1) or the formula (2) and for example, siRNA may be stable under aphysiological condition generally when the N/P ratio is 2 to 60.

The definition N/P ratio=[total number of amino group and substitutedamino group of polycation moiety in side chain]/[total number ofphosphate group in nucleic acid] is applicable.

As for the compound of the formula (1), an optimum N/P ratio variesdepending on the ratio of the hydrophobic group in the total amino groupand hence cannot be specified. In general, however, when a PIC withsiRNA is formed so that the N/P ratio is 5 or more, preferably 7 ormore, a stable associate having an average particle diameter size ofabout one hundred and several tens nm can be provided under aphysiological condition such as in circulating blood. Such PIC can beprepared by mixing the compound of the formula (1) and siRNA in anaqueous solution buffered as necessary so as to achieve the N/P ratio.When the block copolymer of the formula (2) is used, and in particular,the ratio of a hydrophobic group is increased, there is a tendency thatsuch copolymer alone associates autonomously in an aqueous solution toform a polymeric micelle. Thus, a stable PIC can be formed at a widerrange of N/P ratios as compared to the case of using the compound of theformula (1).

Hereinafter, the present invention is further described by way ofspecific examples. However, these examples are provided for illustrativepurposes only.

PRODUCTION EXAMPLE 1 Synthesis of N-succinimidyl octadecanoate

N-Succinimidyl octadecanoate was synthesized in accordance with thefollowing article [N. M. Howarth, W. E. Lindsell, E. Murray, P. N.Preson, Tetrahedron 61 (2005) 8875-8887.]. Stearic acid (1.87 g, 6.56mmol) and N-hydroxysuccinimide (0.76 g, 6.56 mmol) were dissolved in 80mL of dichloromethane (DCM) and subjected to a reaction with1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSC) (1.25g, 6.56 mmol) for 48 hours. The reaction product was then washed withwater, extracted with DCM twice, and dried over MgSO₂ to afford a whitepowder (1.4 g, 56% yield). The conversion rate of a carboxyl group instearic acid was calculated by ¹H-NMR and found to be 96%.

EXAMPLE 1 (1) Synthesis of stearoyl group-introduced PAsp(DET)(PAsp(DET)-ST)

β-Benzyl-L-aspartate-N-carboxylic anhydride (BLA-NCA) was subjected tocleavage polymerization using n-butylamine as an initiator to synthesizepoly(β-benzyl-L-aspartate) (PBLA) (polymerization degree: 110). Next,PBLA (513 mg) was lyophilized from benzene and then dissolved in 25 mLof N-methyl-2-pyrrolidone (NMP). A 50-fold equivalent ofdiethylenetriamine (DET) with respect to a PBLA side chain was mixedwith 13.5 mL of NMP. Then, under argon at 10° C., the DET solution wasadded to the PBLA solution and the mixture was subjected to a reactionfor 1 hour. After that, the resultant was added to a cooled 0.01 N HClaqueous solution, dialyzed against 0.01 N HCl twice, dialyzed againstwater at 4° C., and then collected by lyophilization. Confirmation hasbeen made that PAsp(DET) (on the left of FIG. 1) thus synthesized has ahigh buffer ability while having a cell membrane damaging activity inresponse to a low pH environment, and can transport plasmid DNA into thecytoplasm efficiently (see Non Patent Document 2). Next, PAsp(DET) (100mg, 0.366 mmol in terms of the molar number of an Asp(DET) unit) andDIPEA (638 ml, 3.66 mmol) were dissolved in 6 mL of methanol andsubjected to a reaction with N-succinimidyl octadecanoate (27.8 mg,0.073 mmol) dissolved in 6 mL of DCM at 4° C. for 24 hours. After thereaction, the reaction product was reprecipitated in diethyl ether andfiltered. The resultant sample was dissolved in methanol/water (1:1 v/v)and then dialyzed at 4° C. against a 0.01 M HCl aqueous solution threetimes and distilled water once. After that, the resultant was collectedby lyophilization to synthesize PAsp(DET)-ST having a stearoyl groupintroduced into a side chain (on the right of FIG. 1). The yield of thepolymer was 90% or more.

(2) Complex Formation of PAsp(DET)-ST and siRNA

PAsp(DET)-ST having a stearoyl group introduction rate of 0, 12, or 19%was dissolved in a 10 mM HEPES buffer (pH 7.3) or a 50% ethanol solution(ethanol/10 mM Hepes buffer, 1:1 v/v) and mixed with siRNA (20 μM, 10 mMHEPES buffer (pH 7.3)) at different N/P ratios. Complex formation wasconfirmed by polyacrylamide electrophoresis (FIG. 2 illustrates theresults of electrophoresis of complexes of PAsp(DET)-ST 19% and siRNA).As a result, in the case of using any polymer, a band of free siRNAdisappeared at an N/P ratio equal to or more than a specific value (N/Pratio of 2.5 or more in the case of PAsp(DET)-ST 19%) , suggesting thatthe complex formation of siRNA with PAsp(DET) or PAsp(DET)-ST occurred.Next, PAsp(DET)-ST/siRNA was evaluated for its particle diameter andpolydispersity index (PDI) by dynamic light scattering (Dynamic lightscattering Zetasizer (Malvern Instruments, Worcestershire, U.K.)) (FIG.3). As a result, a large aggregate having a size of 200 nm or more wasformed at a relatively low N/P ratio (N/P ratio of 1.4 to 2.5 in thecase of PAsp(DET) and N/P ratio of 2.0 to 3.5 in the case ofPAsp(DET)-ST), whereas a relatively monodispersed associate having asize of 150 to 170 nm was formed only in the case of PAsp(DET)-ST at anN/P ratio of 3.5 to 4.0 or more. On the other hand, in the case ofPAsp(DET), scattered light intensity was weak and no associate formationwas confirmed.

The above-mentioned results suggest that the introduction of a stearoylgroup as a hydrophobic group into a PAsp(DET) side chain led to complexstabilization through a hydrophobic interaction and nanoparticleformation.

EXAMPLE 2 (1) Knockdown of Endogenous Gene Bcl-2 with PAsp(DET)-ST/siRNAComplex

Human pancreatic cancer Panc-1 cells were seeded in a 6-well plate(100,000 cells/well) and were cultured overnight. Then, a complex ofsiRNA directed against Bcl-2 and each of PAsp(DET) and PAsp(DET)-ST wasformed as described above and cultured with the cells for 48 hours(siRNA concentration: 100 nM). After that, the cells were collected bytrypsinization and total RNA was collected with an Rneasy Mini kit(Qiagen). Next, cDNA was prepared using a TAKARA PrimeScript RT reagentkit and the mRNA amounts of Bcl-2 and GADPH (housekeeping gene as aninternal standard) were evaluated by Real time PCR (ABI7500 FastReal-Time PCR System (Applied Biosystems, Foster City, Calif., USA))using a QuantiTect SYBR Green PCR kit (Qiagen). Here, the followingsequence was used as the siRNA directed against Bcl-2 (5′-CAG GAC CUCGCC GCU GCA GAC-3′; 3′-CGG UCC UGG AGC GGC GAC GUC UG-5′) (Reference 1).The results revealed that PAsp(DET)-ST 12% and PAsp(DET)-ST 19% wereable to knock down mRNA of Bcl-2, which was an endogenous gene and wasapoptosis-suppressive, at an N/P ratio of 7.5 or more and at any N/Pratio, respectively (FIG. 4). Further, non-specific cytotoxicity on thePanc-1 cells due to the siRNA transfection (culture for 40 hours) wasevaluated using a Cell Counting Kit-8 (Dojindo, Japan). Asa result, nocytotoxicity was observed at any stearoyl group introduction rate andN/P ratio (FIG. 5).

(2) Knockdown of Endogenous Gene VEGF with PAsp(DET)-ST/siRNA Complex

Complexes prepared by mixing PAsp(DET)-ST 19% and siRNA at different N/Pratios were cultured with human cervical cancer HeLa cells for 48 hours,and the mRNA amount of vascular endothelial growth factor (VEGF) wasquantitatively determined by Real time PCR (using actin as an internalstandard) in the same manner as described above (FIG. 6). Here, thesequence of siRNA directed against VEGF used was as described below (GGAGUA CCC UGA UGA GAU CdTdT; GAU CUC AUC AGG GUA CUC CdTdT). As a result,PAsp(DET)-ST 19% significantly knocked down the expression of VEGF atany N/P ratio, and the activity was similar to that of Lipofectamine2000 used as a positive control (FIG. 6). On the other hand, PEI (ExGen)did not give any significant gene knockdown effect (FIG. 6). Next, theexpression amount of VEGF at a protein level was evaluated by an ELISA(sandwich immunoassay kit (DVE00, Quantkine® human VEGF, R&D Systems,Minneapolis, Minn.)). The results revealed that PAsp(DET)-ST 19%suppressed the expression amount of VEGF in a sequence-specific mannerat a protein level as well (FIG. 7).

EXAMPLE 3 (1) Synthesis of PEG-SS-PAsp(DET)-ST

PEG-SS-PAsp(DET) in which polyethylene glycol (PEG) and PAsp(DET) werelinked through a disulfide (SS) bond undergoing cleavage in a livingbody was synthesized in accordance with Reference 2. Specifically,BLA-NCA was subjected to ring-opening polymerization using PEG-SS-NH₂ asan initiator in methylene chloride to afford PEG-SS-PBLA having a PBLApolymerization degree of 59 and 98. Next, PEG-SS-PBLA was dissolved inNMP and subjected to a reaction with DET at 5° C. for 40 minutes. Afterthat, the reaction product was neutralized with a 25% acetic acidaqueous solution, dialyzed against 0.01 N HCl at 4° C., and furtherlyophilized to synthesize 0.27 g of PEG-SS-PAsp(DET).

Next, an introduction reaction of a stearoyl group into a PAsp(DET) sidechain of PEG-SS-PAsp(DET) was carried out. The introduction reaction ofa stearoyl group was carried out by a reaction with N-succinimidyloctadecanoate in the same manner as in Example 1. Here, PEG-SS-PAsp(DET)polymers having PAsp(DET) polymerization degrees of 59 and 98,respectively, were subjected to a reaction with N-succinimidyloctadecanoate in an amount corresponding to 20% of a side chain primaryamino group. As a result, PEG-SS-PAsp(DET)-ST polymers having stearoylgroups introduced into 17% and 19% of the side chain, respectively, weresynthesized. Those polymers were dissolved in a 10 mM HEPES buffer (pH7.3) and mixed with siRNA so as to achieve N/P=5.0 to form complexes.The thus formed complexes are each conceivable to have a polymer micellestructure in which a PAsp(DET)-ST/siRNA complex is covered with abiocompatible PEG outer envelope. In addition, PEG is gradually detachedin a living body and thus both of high stability in blood and efficientsiRNA introduction in a target tissue are conceivable to besimultaneously achieved in siRNA delivery through systemicadministration (FIG. 8).

(2) Evaluation of Intracellular Uptake of PEG-SS-PAsp(DET)-ST/siRNAComplex

A PEG-SS-PAsp(DET)-ST/siRNA complex (N/P=5.0) is estimated to have abilayer structure in which a surface is covered with a PEG outerenvelope, and hence has a possibility of undergoing an intracellularuptake different from a PAsp(DET)-ST/siRNA complex having a cationicsurface. In view of the foregoing, a complex was formed using siRNAlabeled with a fluorescent dye Cy5 and evaluated for its intracellularuptake amount. HeLa cells (100,000 cells/well) were cultured on a 6-wellmultiplate and cultured with PAsp(DET)-ST/siRNA complex andPEG-SS-PAsp(DET)-ST/siRNA complex for 3 hours. After that,trypsinization was carried out and the average fluorescence intensity ofCy5 taken up into the cells was evaluated by flow cytometry (BD™ LSR IIflow cytometer). Table 1 shows the results.

TABLE 1 siRNA PEG(10k)-SS-PA PEG(10k)-SS-PA concentration PAsp (DET)-STsp(DET)(59)-ST sp(DET)(98)-ST 100 nM 9215.47 1688.86 1265.89 300 nM6079.30 4542.10 500 nM 13935.21 7071.59

The results revealed that, when the siRNA concentration was 100 nM, theintracellular uptake amount of the PEG-SS-PAsp(DET)-ST/siRNA complex waslower than that of PAsp(DET)-ST/siRNA, but the PEG-SS-PAsp(DET)-ST/siRNAcomplex was taken up into the cells in an amount similar to that ofPAsp(DET)-ST/siRNA by increasing the concentration of the complex.

(3) siRNA Transfection with PEG-SS-PAsp(DET)-ST/siRNA Complex

Human hepatic cancer Huh-7 cells were seeded into a 24-microwell plate(20,000 cells/well) and cultured overnight. After that, plasmid DNA thatcan express Firefly luciferase and Renilla luciferase was introducedinto the cell using Lipofectamine 2000 (Invitrogen). After 4 hours, eachof siRNA complexes formed from PAsp(DET)-ST 19%, PEG-SS-PAsp(DET)(59)-ST 17%, and PEG-SS-PAsp(DET) (98)-ST 19% each carrying siRNAdirected against Firefly luciferase was added to a medium and culturedfor 48 hours. After that, gene knockdown efficiency with siRNA wascalculated based on the expression amount of Firefly luciferase toRenilla luciferase using a Dual-Luciferase Reporter Assasy Kit(Promega). As a result, in 100 nM siRNA, the PEG-SS-PAsp(DET)-ST/siRNAcomplex did not exhibit any significant gene knockdown effect, whereasin 300 and 500 nM siRNA, the complex exhibited a concentration-dependentgene knockdown activity (FIG. 9). The results are in good consistentwith the results of an intracellular uptake in Table 1. Thus, a complexhaving a polymer micelle structure is conceivable to be taken up intocells and then efficiently escaped from the endosome, which allows afunction of siRNA to be efficiently expressed in the cytoplasm.

REFERENCE 1

-   M. Ocker, D. Neureiter, M. Lueders, S. Zopf, M. Ganslmayer, E. G.    Hahn, C. Herold, D. Schuppan, Gut 54: 1298-1308 (2005)

REFERENCE 2

-   S. Takae, Y. Akiyama, Y. Yamasaki, Y. Nagasaki, K. Kataoka,    Colloidal Au replacement assay for highly sensitive quantification    of low molecular weight analytes by surface plasmon resonance.    Bioconjugate Chem. 18(4): 1241-1245 (2007)

1. A cationic poly(amino acid) of formula (1):

wherein: R¹ represents a hydroxyl group, an unsubstituted or substitutedlinear or branched alkyloxy group having 1 to 12 carbon atoms, anunsubstituted or substituted linear or branched alkenyloxy group having2 to 12 carbon atoms, an unsubstituted or substituted linear or branchedalkynyloxy group having 1 to 12 carbon atoms, or an unsubstituted orsubstituted linear or branched alkyl-substituted imino group having 1 to12 carbon atoms; R² represents a hydrogen atom, an unsubstituted orsubstituted linear or branched alkyl group having 1 to 12 carbon atoms,or an unsubstituted or substituted linear or branched alkylcarbonylgroup having 1 to 24 carbon atoms; R^(3a) and R^(3b) each independentlyrepresent a methylene group or an ethylene group; R^(4a) and R^(4b) areeach independently selected from the group consisting of:—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i);—NH—(CH₂)_(p2)—N[—(CH₂)_(q2)—NH₂]₂   (ii);—NH—(CH₂)_(p3)—N{[—(CH₂)_(q3)—NH₂][—(CH₂)_(q4)—NH]_(r2)H}  (iii); and—NH—(CH₂)_(p4)—N{—(CH₂)_(q5)—N[—(CH₂)_(q6)—NH₂]₂}₂   (iv), wherein: p1to p4, q1 to q6, and r1 and r2 each independently represent an integerof 1 to 5; 5 to 40% of a total number of the group of each of R^(4a) andR^(4b) have at least one amino group in which a hydrogen atom issubstituted by an acyl group having a saturated or unsaturated linear orbranched aliphatic hydrocarbon residue having 6 to 27 carbon atoms, or asteroloxycarbonyl group; n represents an integer of 30 to 5,000; and yrepresents an integer of 0 to 5,000, provided that, when R^(3a) andR^(3b) each represent a methylene group, y represents an integer smallerthan n.
 2. The poly(amino acid) of claim 1, wherein R^(3a) and R^(3b)each independently represent a methylene group in formula (1).
 3. Thepoly(amino acid) of claim 1, wherein R^(3a) represents an ethylene groupand y represents 0 in formula (1).
 4. The poly(amino acid) of claim 1,wherein: R^(4a) and R^(4b) each independently represent a group:—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i), wherein: p1 and q1 eachindependently represent 2 or 3; and r1 represents an integer of 1 to 3;and 10 to 30% of a total number of the group of each of R^(4a) andR^(4b) have an amino group in which a hydrogen atom is substituted by anacyl group having a saturated or unsaturated linear or branchedaliphatic hydrocarbon residue having 6 to 27 carbon atoms.
 5. Thepoly(amino acid) of claim 3, wherein: R^(4a) represents a group:—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i), wherein: p1 and q1 eachindependently represent 2 or 3; and r1 represents an integer of 1 to 3;and 10 to 30% of a total number of the group of R^(4a) have an aminogroup in which a hydrogen atom is substituted by an acyl group having asaturated or unsaturated linear or branched aliphatic hydrocarbonresidue having 6 to 27 carbon atoms.
 6. A block copolymer of formula(2):

wherein: R² represents a hydrogen atom, an unsubstituted or substitutedlinear or branched alkyl group having 1 to 12 carbon atoms, or anunsubstituted or substituted linear or branched alkylcarbonyl grouphaving 1 to 24 carbon atoms; R^(3a) and R^(3b) each independentlyrepresent a methylene group or an ethylene group; R^(4a) and R^(4b) areeach independently selected from the group consisting of:—NH—(OH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i);—NH—(CH₂)_(p2)—N[—(CH₂)_(q2)—NH₂]₂   (ii);—NH—(CH₂)_(p3)—N{[—(CH₂)_(q3)—NH₂][—(CH₂)_(q4)—NH]_(r2)H}  (iii); and—NH—(CH₂)_(p4)—N{—(CH₂)_(q5)—N[—(CH₂)_(q6)—NH₂]₂}₂   (iv), wherein: p1to p4, q1 to q6, and r1 and r2 each independently represent an integerof 1 to 5; 5 to 40% of a total number of the group of each of R^(4a) andR^(4b) have at least one amino group in which a hydrogen atom issubstituted by an acyl group having a saturated or unsaturated linear orbranched aliphatic hydrocarbon residue having 6 to 27 carbon atoms, or asteroloxycarbonyl group; n represents an integer of 30 to 5,000; and yrepresents an integer of 0 to 5,000, provided that, when R^(3a) andR^(3b) each represent a methylene group, y represents an integer smallerthan n; L¹ represents —S—S— or a valence bond; L² represents —NH—, —O—,—O(CH₂)_(p1)—NH—, or -L^(2a)-(CH₂)_(q1)-L^(2b)-, wherein: p1 and q1 eachindependently represent an integer of 1 to 5; L^(2a) represents OCO,OCONH, NHCO, NHCOO, NHCONH, CONH, or COO; and L^(2b) represents NH or O;R⁵ represents a hydrogen atom or an unsubstituted or substituted linearor branched alkyl group having 1 to 12 carbon atoms; and m represents aninteger of 30 to 20,000.
 7. A polyion complex, comprising: thepoly(amino acid) of claim and a nucleic acid.
 8. A polyion complex,comprising: the block copolymer of claim 6; and a nucleic acid.
 9. Thecomplex of claim 7, wherein the nucleic acid is selected from the groupconsisting of plasmid DNA, siRNA, micro RNA, an antisense nucleic acid,a decoy nucleic acid, an aptamer, and a ribozyme.
 10. The complex ofclaim 8, wherein the nucleic acid is selected from the group consistingof plasmid DNA, siRNA, micro RNA, an antisense nucleic acid, a decoynucleic acid, an aptamer, and a ribozyme.
 11. The poly(amino acid) ofclaim 2, wherein: R^(4a) and R^(4b) each independently represent agroup:—NH—(CH₂)_(p1)—[NH—(CH₂)_(q1)—]_(r1)NH₂   (i), wherein: p1 and q1 eachindependently represent 2 or 3; and r1 represents an integer of 1 to 3;and 10 to 30% of a total number of the group of each of R^(4a) andR^(4b) have an amino group in which a hydrogen atom is substituted by anacyl group having a saturated or unsaturated linear or branchedaliphatic hydrocarbon residue having 6 to 27 carbon atoms.
 12. Thepoly(amino acid) of claim 1, wherein R^(4a) and R^(4b) are the samegroup.
 13. The poly(amino acid) of claim 1, wherein p1 to p4 and q1 toq6 each independently represent 2 or 3
 14. The poly(amino acid) of claim1, wherein p1 to p4 and q1 to q6 each represent
 2. 15. The poly(aminoacid) of claim 1, wherein r1 and r2 each independently represent aninteger of 1 to
 3. 16. The poly(amino acid) of claim 1, wherein R¹represents a hydroxyl group.
 17. The poly(amino acid) of claim 1,wherein R¹ represents an unsubstituted or substituted linear or branchedalkyloxy group having 1 to 12 carbon atoms.
 18. The poly(amino acid) ofclaim 1, wherein R¹ represents an unsubstituted or substituted linear orbranched alkenyloxy group having 2 to 12 carbon atoms.
 19. Thepoly(amino acid) of claim 1, wherein R¹ represents an unsubstituted orsubstituted linear or branched alkynyloxy group having 1 to 12 carbonatoms.
 20. The poly(amino acid) of claim 1, wherein R¹ represents anunsubstituted or substituted linear or branched alkyl-substituted iminogroup having 1 to 12 carbon atoms.